Letters to the Editor 229 Figure 1 (a–d) The BCR–ABL variants show similar tyrosine phosphorylation profiles in vitro.(a) Domain organization of the three BCR–ABL variants tested in this study. The coiled-coil (CC), DBL and pleckstrin homology (PH) domains pertain to BCR; the SH3, SH2 and TK domains pertain to ABL. The p200BCR–ABL variant lacks the PH domain, while p190BCR–ABL lacks both the PH and DBL-like domains. Each BCR–ABL variant was stably expressed in 32D cells and whole-cell lysates were subjected to immunoblot analysis with antibodies specific for (b) ABL or (c) global tyrosine phosphorylation (4G10). (d) Immunoblot analysis of STAT5 and STAT6 phosphorylation in whole cell lysates from Ba/F3 cells expressing each of the three BCR–ABL variants. (e–h) Deletion of the PH domain is sufficient to increase the lymphoid transformation potency of BCR–ABL. Donor bone marrow cells from (e) 5-FU-treated Balb/c mice or (f) non-5-FU-treated Balb/c mice were infected with equivalent titer retrovirus expressing p210BCR–ABL, p200BCR–ABL or p190BCR–ABL and transplanted into lethally irradiated recipient mice. Kaplan–Meier survival curves are shown. Mortality events due to CML-like disease, B-ALL-like disease and unrelated causes are marked with square, circle and triangle symbols, respectively. (g, h) Representative fluorescence-activated cell sorting analyzes for CML-like and B-ALL-like disease, respectively. Peripheral blood cells were measured for GFP expression on the y axis. The x axis represents staining with antibodies specific for myeloid cells (Ly6G), B cells (B220), T cells (Thy1.2), early myeloid cells (CD11b) or erythroid cells (Ter119). CML-like and B-ALL-like disease was analyzed in different mouse tissues using GFP (y axis) and either Ly6G or B220 (x axis), respectively. a slight further increase is observed with the additional deletion 2Howard Hughes Medical Institute, Portland, OR, USA of BCR exons 2–8. This is consistent with the data presented by E-mail: [email protected] Li et al., which showed that p190BCR–ABL but not p210BCR–ABL induced B-ALL in this model. Given that the associations References between the type of BCR–ABL protein and the disease 7 phenotype are not stringent, additional mechanisms must be 1 Deininger MW, Goldman JM, Melo JV. The molecular biology of operational. For example, pre-B cells may have a higher chronic myeloid leukemia. Blood 2000; 96: 3343–3356. likelihood of acquiring 190BCR–ABL compared with hemato- 2 Li S, Ilaria Jr RL, Million RP, Daley GQ, Van Etten RA. The P190, poietic stem cells that are thought to be the origin of CML. P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have Although it remains to be established that PH domain- different lymphoid leukemogenic activity. J Exp Med 1999; 189: dependent signals are critical to phenotype selection, our results 1399–1412. show a role for this domain in BCR–ABL signal transduction and 3 Lugo TG, Pendergast AM, Muller AJ, Witte ON. Tyrosine kinase transforming capacity. activity and transformation potency of bcr-abl oncogene products. Science 1990; 247: 1079–1082. 4 Kin Y, Li G, Shibuya M, Maru Y. The Dbl homology domain of Conflict of interest BCR is not a simple spacer in P210BCR-ABL of the Philadelphia chromosome. J Biol Chem 2001; 276: 39462–39468. 5 Ilaria Jr RL, Van Etten RA. P210 and P190(BCR/ABL) induce The authors declare no conflict of interest. the tyrosine phosphorylation and DNA binding activity of multi- ple specific STAT family members. J Biol Chem 1996; 271: 31704–31710. Acknowledgements 6 Benekli M, Baer MR, Baumann H, Wetzler M. Signal transducer and activator of transcription proteins in leukemias. Blood 2003; This study was supported in part by the Leukemia and Lymphoma 101: 2940–2954. Society (BJD, MWD), the TJ Martell Foundation (BJD) and NHLBI 7 Melo JV. The diversity of BCR-ABL fusion proteins and their grant HL082978-01 (MWD). JWT is supported by the William relationship to leukemia phenotype. Blood 1996; 88: 2375–2384. 8 Russo C, Gao Y, Mancini P, Vanni C, Porotto M, Falasca M et al. Lawrence Foundation and the Oregon Clinical and Translational Modulation of oncogenic DBL activity by phosphoinositol Research Institute. MWD is a Scholar in Clinical Research of the phosphate binding to pleckstrin homology domain. J Biol Chem Leukemia and Lymphoma Society. BJD is an investigator of the 2001; 276: 19524–19531. Howard Hughes Medical Institute. 9 Han J, Luby-Phelps K, Das B, Shu X, Xia Y, Mosteller RD et al. Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 1998; 279: S Demehri1, T O’Hare1,2, CA Eide1,2, CA Smith1, JW Tyner1, 1,2 1 558–560. BJ Druker and MWN Deininger 10 Rossman KL, Worthylake DK, Snyder JT, Siderovski DP, Campbell 1 Division of Hematology and Medical Oncology, Oregon SL, Sondek J. A crystallographic view of interactions between Dbs Health and Science University Knight Cancer Institute, and Cdc42: PH domain-assisted guanine nucleotide exchange. Portland, OR, USA and Embo J 2002; 21: 1315–1326. Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu) Inhibition of Syk protein tyrosine kinase induces apoptosis and blocks proliferation in T-cell non-Hodgkin’s lymphoma cell lines Leukemia (2010) 24, 229–232; doi:10.1038/leu.2009.198; prognosis compared with aggressive B-cell lymphomas. There- published online 24 September 2009 fore, novel targeted therapies are needed. Syk is a protein tyrosine kinase involved in B-cell receptor signaling in both Peripheral T-cell lymphomas (PTCLs) are among the most normal and malignant B cells.1 Syk activation in B-cell aggressive of all lymphomas and are associated with a poor lymphomas is associated with cell growth and survival, and an Leukemia Letters to the Editor 230 orally available Syk inhibitor is currently being tested for B-cell To determine whether Syk contributes to the growth and lymphomas in a phase I/II clinical trial, with encouraging survival of T-cell lymphomas, a small interfering RNA (siRNA) results.2 Normal T lymphocytes lack Syk expression and use a approach was used to inhibit Syk expression. Syk siRNA Syk homolog, Zap-70, in receptor signaling. However, we effectively silenced Syk expression in both SU-DHL-1 and recently showed that Syk protein is aberrantly expressed in the SR-786 cells (Figure 2a). As Syk inhibition blocks proliferation majority of PTCLs.3 The functional role of Syk in PTCL is and induces apoptosis in B-cell lymphomas,1 we determined the currently unknown. Herein, we show that silencing Syk induces effects of Syk siRNA on PTCL cells. In both SU-DHL-1 and SR- apoptosis and blocks proliferation in PTCL cells. These findings 786, Syk siRNA significantly reduced both proliferation (3H-TdR suggest that Syk represents a novel therapeutic target for patients incorporation, Figure 2b) and cell viability (annexin V assay, with PTCL. Figure 2c). Syk siRNA also silenced Syk expression in SeAx cells, To establish an in vitro model for examining the effects of Syk in which Syk is not phosphorylated (data not shown). Prolifera- inhibition, we analyzed Syk expression in multiple PTCL cell tion of SeAx cells was determined after treatment with either a lines (SeAx, HuT 78, SR-786, Karpas 299 and SU-DHL-1) by control or Syk siRNA in a manner identical to that described in both western blot (Figure 1a) and flow cytometry (Figure 1b). Figure 2b. No significant difference in proliferation (67 691 þ /À Consistent with our previously reported data showing Syk 1974 cpm versus 73 884 þ /À 3467 cpm, respectively; n ¼ 3) expression in PTCL, three of five cell lines tested (SU-DHL-1, was observed between the two treatment groups. SR-786 and SeAx) showed strong Syk expression.3 Next, we As siRNA-based inhibition of therapeutic targets is difficult to determined the phosphorylation status of Syk in these cell lines. achieve clinically, we determined whether a Syk inhibitor currently SU-DHL-1 and SR-786 cells, but not SeAx cells, showed in clinical trial, R406,2 would recapitulate the functional effects of Syk phosphorylation at Y348 (Figure 1c and d), a site autopho- Syk siRNA in PTCL cells. As expected, R406 led to depho- sphorylated on B-cell receptor engagement in B cells.4 Syk was sphorylation of Syk in both SU-DHL-1 and SR-786 (Figure 2d). also phosphorylated at Y525/526 in both SU-DHL-1 and SR-786 Furthermore, R406 significantly inhibited cell proliferation in a (data not shown). dose-dependent manner (Figure 2e), and induced cell death in Figure 1 Syk is expressed and phosphorylated in T-cell lymphomas. Cell lines (anaplastic large cell lymphoma cell lines SU-DHL-1, SR-786 and Karpas 299, and the cutaneous T-cell lymphoma cell lines SeAx and HuT 78 were used, as indicated) were analyzed for total Syk expression by western blot (a) and flow cytometry (b). (a) Protein lysates prepared from cell lines were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) and incubated with primary antibodies to Syk or b-actin (BD, Franklin Lakes, NJ, USA), followed by anti-mouse secondary antibody (Pierce, Rockford, IL, USA). (b) Fluorochrome-conjugated antibodies (BD) were used to determine Syk expression using intracellular staining and flow cytometry on a FACSCalibur instrument (BD) as per the manufacturer’s recommendations. CellQuest or FACSDiva Software (BD) were used for analysis. (c) Similarly, intracellular phospho-specific flow cytometry was used to assess Syk (Y348) phosphorylation.